1. Field of the Invention
This invention relates generally to apparatus and method for performing a molecular biological reaction, e.g., a polymerase chain reaction (PCR) process. More particularly, this invention relates to an apparatus and method for creating a sealed space by a simplified method and apparatus for performing an in situ PCR on a DNA or RNA sample in the original cell structure by localizing the PCR reaction mixture within a resin circled space on slides to carry out the PCR process on nucleic acid sample such that the PCR process can be more flexibly performed at a lower cost.
2. Description of the Prior Art
For an in situ polymerase chain reaction (PCR), due to the requirement of conducting the thermal cycling processes in a specially sealed space on slides with suitable conditions for DNA amplification, current techniques are still restricted by a limitation that the more costly containment systems specially designed for in situ PCR have to be used in order to satisfy this requirement. Other than the fact the PCR containment systems are more expensive, such containment systems are also inflexible due to its rigid structure. Due to their rigid structure, for those of ordinary skill in the art, under certain circumstances the conventional containment systems are more difficult to apply. Additionally, the sealed space provided by the containment system with rigid structure is fixed and cannot be flexibly changed. As the result of this inflexibility, additional economic penalty may incur under the circumstances in amplifying small volumes of target cells or tissues. Due to the fact that the sealed space is fixed and cannot be reduced, despite the samples are of smaller volume, same amount of enzyme and reagents are still required to fill the sealed space to assure complete amplification processes are performed. Thus the PCR containment systems of rigidly structured as now available causes unnecessary wastes of expensive enzyme and reagents and leads to even higher cost in conducting the PCR amplification process. On the contrary, when the samples are of larger size than the containment systems, due to the fixed size limitation, the in situ PCR amplifications cannot be performed with the containment system unless the samples are further processed for volume reduction.
A segment of double stranded DNA can be amplified by applying a polymerase chain reaction (PCR) process wherein a very large number of faithful copies of the template DNA can be produced. The PCR process is carried out by thermal-cycling of the template DNA in the presence of thermally stable DNA polymerase enzyme which typically is a Taq polymerase, four DNA nucleotide bases and two or more single stranded DNA primers. The PCR techniques are disclosed in several prior U.S. Patents and are commonly practiced by those of ordinary skill in the art of molecular biology and other related fields. Recent advancement in PCR technology enables the PCR processes to be carried out on specific DNA segments inside cells without first extracting DNA from the cells. This in situ PCR processes are typically performed on sample tissues or cells which are fixed by treatment with formalin to be mounted on microscopic slides with the morphology of the sample tissues or cells preserved for recognition after treatment. Thermal cycling are then performed on a mixture of the sample tissues or cells including the target DNA and developer reagents and other PCR reagents attached with label-molecule or DNA-probe also attached with a label molecule. After the post-PCR processing steps are performed after the in situ PCR thermal cycling is completed, a multiple, e.g., 10.sup.6 to 10.sup.12, copies of the amplified DNA segments in each cell are generated.
During the thermal cycling process, the specimen for amplification covered with PCR reagents containing DNA polymerase enzyme, nucleotides, primers and other components are mounted on a silanized slide. The slide and the reagent mixture are cycled typically from about 50.degree. C. to 95.degree. C. for approximately 20 to 30 times. In order to assure proper conditions for DNA amplification, the concentration of the reagent mixture must be carefully maintained. Prevention of water evaporation from the reagent mixture is required to maintain an optimum reagent concentration such that the PCR operation can be successfully carried out.
Several techniques are being applied to provide a sealed space to carry out the PCR thermal cycling process therein. An early method is to place a cover slip over a sample for amplification and then seal it with nail polish or similar adhesive material. Leakage often occurs in this type of arrangement as the seal provided by the nail polish does not adhere properly to the cover slip. Furthermore, the nail polish seal often cannot sustain the temperature and pressure required for the PCR operation and resulting in a dislodge of the seal thus causing the cover slip to touch the cells and damaging the cell morphology. A more complicated post-PCR process is also required to dissolve the nail polish with a chloroform treatment.
Other techniques involving the application of chambered slides enclosed by gasket and covered with Saran wrap; placing the reagents in an aluminum boat then covered it with polypropylene. Adding the mineral oil on top of the PCR mixture sample to reduce evaporation and condensation is also applied in different PCR amplification processes. However, these techniques rely heavily on the skill of the persons who perform these operations to minimize leaks, control the reactions in the confined space, and to place the slides and the covers in a right position to achieve the desire results. These techniques may be useful and applicable in small scale, however, their results usually cannot be reproducible and therefore are very limited in their usefulness for broader and routine applications where reliable and reproducible results are required.
Atwood et al. disclose in U.S. Pat. No. 5,364,790, entitled "In Situ PCR Amplification System" (issued on Nov. 15, 1994), an in situ PCR system for amplification of nucleic acids contained in a prepared cell or tissue sample. The PCR system 10 is shown in FIGS. 1A and 1B. The PCR amplification system 10 is a containment includes a glass microscope slide 14, a specimen sample 12 containing the target nucleic acid sequence mounted on the slide 14, a flexible plastic cover 16 over the sample 12 and a retaining assembly 28 fastened to the slide 14 and to the cover 16 to retain and seal a reaction mixture 13 against the sample 12 during thermal cycling. The retaining assembly includes a rigid ring 20 on a rim portion 19 of the cover 16, a cross beam 24 having spaced parallel rails joined by opposite flat ends, and a pair of clips 28 which are pressed over the ends and opposite sides of the slide 14 to fasten the cross beam 24 and cover 16 to the slide 14. The cover 16 is retained against the slide 14 and a sealed space is created to contain the reaction mixture during thermal cycling.
Atwood et al. provide a secure and well sealed space for carrying out the PCR amplification process. However, as discussed above, due to the rigid structure of this containment system, difficulties arise in wasting expensive reagents in applying the apparatus to small volumes of samples. As special training is required to follow the operation procedure of the system, a leakage may still occur if specific procedures are not followed carefully. Furthermore, additional costs are involved in applying this technique to PCR amplification due to the fact that the patented containment system, as now marketed and made commercially available, is quite expensive. Also, in order to properly use this system, various consumable items, e.g., clamps, slips, cover slips and particular reagents all have to be purchased from certain suppliers according to the specifications of the containment system. These cost considerations further limit broader and useful PCR applications.
An alternate reaction containment system is provided in a "Winter 1996 Catalog" published by PGC Scientific. As shown in FIG. 2, a removable, self adhesive chamber enclosing a 15 mm diameter specimen area on a microscope slide to control evaporation for in situ DNA amplification during thermal cycling on microscope slides is disclosed by PGC Scientific. The chamber defines a cone shaped space which is composed of materials that is heat set adhesive to securely attach to the slide. A fixed amount of reagents may be added followed by a self-adhesive top seal which is flexible to minimize the internal pressure changes. Even that this cone-chamber made of self adhesive materials are simpler and more convenient to apply than the containment system disclosed by Atwood et al. as now available in the market, however, the chamber is still limited by a fixed volume. In the same Catalog, other types of "chambers" and "wells" are disclosed which employ pre-formed adhesive "Probe-Clip" to provide a sealed space. Again, these types of apparatuses are limited by the rigid structure and fixed volume which is pre-defined by the structure of the chambers or wells. The limitations due to the inflexibility in changing the volumes contained by the sealed space are not resolved by the techniques provided by PGC Scientific.
Therefore, a need still exists in the art of apparatus and operation technique of molecular biological processing to provide an appropriate seal-materials and structural configuration to provide a sealed space suitable for thermal cycling operations and post-thermal cycling treatment in removing the seal materials such that processes involving molecular biological reactions such as PCR can be more economically and flexibly performed.